Mammalian stem cells are undifferentiated, primitive cells which have the ability both to multiply and to differentiate into specific kinds of cells. Embryos provide a high concentration of stem cells and stem cell lines derived from embryos, embryonic stem (ES) cells, are pluripotent, thus possessing the capability of developing into any cell. These cells are immortal and can be maintained in an undifferentiated state in culture or directed to undergo differentiation into extra embryonic or somatic lineages. More recently, it has been recognised that embryonic germ (EG) cells i.e. cells derived from primordial germ cells may have similar properties to ES cells. Other stem cells may be derived from adults and include mesenchymal, epithelial and neural stem cells.
Such stem cells represent a major potential for cell therapies for regenerative medicine as differentiated cells can be generated for transplantation, may be genetically modified and can be transplanted as pure populations or, following tissue engineering, as tissues or physiologically functional parts of organs (organoids). ES cells are also useful models for studying the cellular and molecular biology of early development and functional genomics. In vitro culture of stem cells can also provide a useful system for drug screening and drug discovery. ES cells derived from mouse embryos are routinely used in a number of laboratory techniques ranging from gene knockout studies, for example generating “knock out” mice models, to transplantation therapies (Sato el al. (2001)).
Stem cells are generally difficult to culture in vitro and careful control of culture conditions, including the appropriate quality of serum and culture medium, is require. This is particularly important if such cells are to be genetically modified or manipulated to introduce genetic mutations, to be grown on a large scale or to direct their differentation towards specific cell types. In addition, careful control and analysis of the differentiation status is required to ensure that the cultured stem cells are suited for their particular use. The selection of appropriate starting cells for directing appropriate phenotypic differentiation is essential as failure can lead not only to a lack of benefit but also to significant side-effects which can include proliferation of differentiated cells. In particular, if cells are not fully differentiated at the time of implantation there is always the possibility of tumour formation. It is therefore clearly important to be able to confirm and select for the undifferentiated integrity or differentiation state of cells within a stem cell population.
Some makers of the status of stem cells are known. Markers currently used for analysis of the undiffereniated integrity of ES cells include Oct 3/4 (Rathjen et al. (1999)), Rex-1 (Ben-Susbhan et al. (1998)), the cell-surface Forssman antigen (Willison et al. (1978); Ling et al. (1997)) and alkaine phosphatase (Rathjen et al. (1999)) (Table 1). All these markers are expressed in undifferentiated ES cells and their levels decrease upon differentiation, However, they are not useful for predicting both the undifferentiated integrity and differentiation state of ES cells since they decrease relatively slowly following the onset of differentiation (Lake et al. (2000); Rathjen et al. (1999)). Additionally, with the exception of the Forssman antigen, the analyses are destructive to cells and require relatively large numbers of cells for RNA extraction.
Removal of leukemia inhibitory factor (LIF) from the medium results in mouse ES cell differentiation (Smith et al. (1992)), characterised by the upregulation of transcript markers such as fibroblast growth factor-5 (Fgf-5), zeta globin (ZG) and Flk-1 (Table 3). However, these markers are transiently expressed and present only on a sub-population of cells thereby limiting their use as single assay markers of ES cell integrity and differentiation.
To date, there is no marker that can accurately assess both the undifferentiated integrity and differentiated state of stem cells. Current analyses of these parameters are time-consuming, often destructive to cells, and require several different markers (Weinhold et al. (2000); Lake et al (2000); Rathjen et al. (1999)). Analysis in a single, non-destructive assay would be a valuable tool for a wide range of ES cell techniques (Lake et al. (2000); Thorey et al. (1998); Niwa et al. (2000); Wakayama et al (1999)).
The 5T4 oncofoetal antigen is a 72 kDa highly glycosylated single pass transmembrane glycoprotein originally isolated from human placental trophoblast (Hole, N. & Stern, P. L. (1988); Hole, N. & Stern, P. L. (1990) and Myers, K. A. et al. (1994). 5T4 has been extensively characterised (see, for example, WO 89/07947). It exhibits restricted expression patterns in human adult issues, being expressed by trophoblast and a few specialised adult epithelia, but is upregulated on many carcinomas, with tumour overexpression correlating with poorer clinical outcome in ovarian, gastric and colorectal cancers. (Southall, P. J. et al. (1990); Wrigley, E. et al. (1995); Starzynska, T. et al. (1994); Starzynska, T. et al. (1998); Mulder, W. M. et al. (1997); Starzynska, T. et al. (1992)). The pattern of 5T4 expression in stem cell populations has not previously been identified.